Myocardial ischemia-reperfusion injury is the most common cause of death in the US. In experimental models, relatively young animals can be protected from ischemia-reperfusion injury through activation of protective signaling pathways, but this protective effect is much more difficult to induce in older animals. Since coronary artery disease in patients is primarily a disease of older individuals, experimental cardioprotective interventions may not be applicable to the most relevant human population. In addition, males and females have different sensitivities to ischemia-reperfusion injury, particularly premenopausal females. One signaling pathway that has major importance in cardioprotection is the activation of nitric oxide synthase, which can result in nitrosylation of protein cysteine residues. Premenopausal females develop greater stress-induced protein S- nitrosylation than males or ovariectomized females, which may account for some of the male-female differences in ischemia-reperfusion injury. However, progress in this area has been hampered by the lack of sensitive methods for detecting nitrosylation of specific cysteine residues in specific proteins, and relating this to cardioprotection. One of the benefits of S-nitrosylation (SNO) is that it is reversible and it may block cysteines from other oxidative modifications during ischemia-reperfusion, preventing proteins from being irreversibly oxidized or covalently modified by disulfide formation. To assess the importance of SNO, methods are needed that can specifically detect post-translational modifications (PTMs) of specific cysteine residues in specific proteins. This proposal is to employ recently developed methods that allow both SNO and oxidation of cysteine residues on specific proteins to be measured using a resin binding approach and mass spectrometry. These innovative methods will permit identification of which specific proteins are modified by cardioprotective interventions, whether there is a difference between males, premenopausal females, and ovariectomized females, and whether aging impacts these processes. Glycogen Synthase Kinase-32 (GSK-32) is also implicated as a critical element in many cardioprotective pathways. These could be separate or interconnected mechanisms, and we propose to test whether GSK-32 affects protein SNO, and whether GSK-32 is necessary for SNO-induced cardioprotection, using cardiac specific conditional GSK-32 deficient mice. The specific aims are (1) to determine the mechanisms of cardioprotection associated with protein SNO, (2) to determine how aging affects protein SNO in male hearts, and (3) to determine how age and hormone status affects protein SNO in females. The results of these studies will provide insights into the mechanisms of cardioprotection, the role of SNO in regulating myocardial function, as well as insights into male-female differences and the effects of aging on cardioprotective strategies. Better understanding of these mechanisms should provide guidance for the development and implementation of strategies for minimizing ischemia-reperfusion injury and adjusting treatments depending on age and sex, and for women, whether they are premenopausal or postmenopausal. PUBLIC HEALTH RELEVANCE: Ischemic heart disease is a major cause of death in the US, and occurs more often at older ages in men and in postmenopausal women. There are very effective strategies for protecting the heart against ischemia- reperfusion injury in the young and healthy but these are generally less effective in older men and postmenopausal women, for reasons that are unclear. This proposal will test one important reason for the effect of age and hormone status in women on ischemic heart disease, and may provide a basis for adjusting therapy as a function of age and hormone status in women.